Process for dyeing textiles and enzymes used therein

ABSTRACT

The present invention relates to a process wherein one or more dye precursors, e.g., indole, are provided to a textile and converted by one or more enzymes, e.g., an oxidizing enzyme, to provide the textile with a dye, e.g. indigo. At least the oxidizing enzyme is a hybrid enzyme including a binding domain that is suitable to bind the enzyme to the textile and/or increase the affinity of the enzyme for the textile, in particular, a cellulose binding domain (CBD).

This Non-Provisional Application claims priority to and the benefit ofEuropean Application No. EP20151830.5 filed on 14 Jan. 2020 the contentof which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a process for dyeing textiles, inparticular for dyeing textiles using enzymes.

BACKGROUND OF THE INVENTION

Vat dyes are insoluble dyes that require a reducing agent to besolubilized in water. Conventionally, dyeing with vat dyes includesapplying the dye in its soluble, reduced form to the textiles andsubsequently oxidizing the dye back to the insoluble form, which conferscolor to the textile.

Indigo is a vat dye of Formula I:

Substitutions on the indigo aromatic ring(s) with groups such ashalogen, alkyl, alkoxy, amino, aryl, aryloxy, and carbonyl, providecompounds that span in a wide range of colors other than blue, and arepart of the so-called indigo derivatives.

A large percentage of indigo and indigo derivatives is produced viasynthesis. Heumann synthesis and Pfleger synthesis were the firstsynthetic routes used for indigo manufacturing at industrial scale;variations of these methods are still in use today. The same syntheticroutes are also used to manufacture indigo derivatives.

Synthesis of indigo and derivatives thereof, as well as other vat dyes,can also be carried out by means of enzymes, or by bacteria expressingenzymes.

While precursors of indigo are soluble in aqueous solutions, indigo isnot, and it precipitates after its synthesis in aqueous solutions.Therefore, as said above, indigo, as well as its derivatives, is usuallyreduced to its leuco form, i.e., leuco indigo, which is the reduced,water soluble, form of indigo, to be applied to textiles.

Therefore, industrial dyeing methods using indigo or derivatives thereofas a dye, or using vat dyes in general, comprise treating an aqueoussolution comprising suspended indigo or derivatives thereof withreducing agents to obtain an aqueous solution comprising leuco-indigo(or the leuco form of such derivatives thereof). The aqueous solutioncomprising leuco-indigo is then applied onto textiles. Indigo or indigoderivatives are obtained by oxidation of leuco-indigo or of the leucoform of indigo derivatives on the textile, thus dyeing the textile. Suchoxidation can be carried out, for example, with the oxygen in the air,e.g., by exposing the textile provided with, for example, leuco-indigo,to air. Usually, the indigo dyeing process requires several impregnationand oxidation steps to reach the desired shade of color.

The reduction of, for example, indigo to leuco indigo is particularlyuseful when the textile to be dyed includes a cellulosic material, suchas cellulosic fibers or yarns. In fact, reduction of indigo to its watersoluble leuco form allows the textile to be provided (e.g., impregnated)with the solution including leuco indigo; subsequently, oxidation ofleuco indigo to indigo occurs on the textile.

The reducing agents used to reduce insoluble vat dyes, such as indigo orderivatives thereof, to leuco indigo or the leuco form of indigoderivatives, are harsh chemicals, i.e. hazardous chemicals for usersand/or environment, such as sodium hydroxide and sodium hydrosulfite.Additionally, large quantities of reducing salts and hydroxides are usedin conventional dyeing processes where indigo or derivatives thereof areused as dyes, thus generating great amounts of wastewater that must betreated before being disposed. This step adds to costs of the dyeingprocess.

A further problem with known indigo dyeing process is that the textile,especially cellulose, may be damaged by extended exposure to thealkaline process solution and chemical products therein present.

There is thus a need for an improved method for dyeing textiles with vatdyes, in particular with indigo and derivatives thereof, that allows fora reduction in the use of water and of harsh chemicals, for example thereducing agents, without losing dyeing effectiveness, so that theoverall cost for vat dyeing and of the waste water treatment processes,if required, is reduced.

SUMMARY OF THE INVENTION

Aim of the present invention is to solve the above-mentioned problemsand to provide a process for dyeing textiles that allows for a reductionin the use of harsh chemicals, such as reducing agents, while providingan effective dyeing.

Also aim of the present invention is to provide a process for dyeingtextiles that is safe, cost-effective and environmentally friendly.

Another aim of the present invention is to provide a process for dyeingtextiles that is more sustainable with respect to conventional dyeingmethods.

These and other aims are reached through the present invention thatprovides a process according to claim 1, namely a process for dyeing atextile, wherein a modified enzyme is involved in the synthesis ofindigo and/or indigo derivatives.

The present invention also relates to processes for dyeing textilesaccording to claims 15, 16 and 17, to a dyed textile according to claim18 and to a modified enzyme according to claim 19 and to claim 21. Thepresent invention further relates to a textile comprising one or moremodified enzymes according to claim 22.

Preferred embodiments of the invention are object of dependent claims2-14 and 20.

DETAILED DESCRIPTION

The present invention relates to a process for dyeing textiles, inparticular textiles including a cellulosic material, comprising thefollowing steps:

-   -   a) providing indole or an indole derivative to at least part of        said textile;    -   b) providing at least an oxidizing enzyme to at least part of        said textile, whereby said at least part of textile includes        said indole or indole derivative and said oxidizing enzyme;    -   c) converting at least part of said indole or indole derivative        to indigo or indigo derivative, thereby dyeing at least part of        said textile;        wherein said oxidizing enzyme is an (oxidizing) hybrid enzyme        comprising a cellulose-binding domain (CBD).

According to embodiments, indole is applied to the textile as an indoleprecursor. Namely, step a) comprises a step of providing tryptophan or atryptophan derivative, and at least a tryptophanase, to said textile,and converting said tryptophan or a tryptophan derivative in thepresence of at least a tryptophanase, to obtain said indole or saidindole derivative.

Through the process of the invention, it is possible to dye textilesavoiding, or substantially avoiding, the use of harsh chemicals, whileobtaining an effective dyeing of the textile.

Moreover, through the process of the invention it is possible to provideinsoluble dyes, such as indigo, on a textile in a controlled manner.

In particular, when an oxidizing enzyme comprising a cellulose-bindingdomain, i.e., an oxidizing hybrid enzyme, is used, an effective dyeingof the textile can be obtained. Without being bound to a specificscientific explanation, an oxidizing enzyme that has been geneticallymodified to include a CBD has an increased affinity for bindingcellulosic fabrics or textiles, with respect to the unmodified oxidizingenzyme. When using the modified enzyme, the conversion of indole (or anindole derivative) to indoxyl (or an indoxyl derivative) and thesubsequent conversion of indoxyl (or an indoxyl derivative) to indigo(or indigo derivative) occur on the textile, whereby effectively dyeingthe textile.

The use of a modified oxidizing enzyme including a CBD according to theinvention allows for providing to the textile an increased amount ofdye, in particular on the surface of the textile material (thusobtaining a ring-dyeing effect), with respect to the use of theunmodified oxidizing enzymes.

According to an aspect, the process of the present invention allows theproduction of dyed textiles. Dyed textiles obtainable through theprocess of the invention may have a variety of colors. In fact,advantageously, by varying the reagents (e.g., indole or a derivativethereof) in the process of the invention, different dyes, may beobtained, so that different final colors can be imparted to textiles.

Also, reagents suitable to be used in the process of the inventionincluding waste water treatment are not expensive, so that the processof the invention is particularly cost-effective with respect to thecurrently available dyeing processes.

In the following description, “textile(s)” and “cellulosic textile(s)”refer to any fiber(s), yarn(s), rope(s), fabric(s) and garment(s) thatinclude cellulosic materials and/or fibers.

As used herein, the terms “cellulose material” and “cellulosic material”refer to any cellulose-containing material. Suitablecellulose-containing materials, i.e., cellulosic materials, are e.g., inthe form of filaments or fibers, such as in cotton, ramie, jute, flax(linen), viscose, rayon, modal, lyocell (Tencel™), bamboo, and mixturesthereof. Suitable cellulosic material may be also obtained frommicroorganisms, for example, by culturing microorganisms that producecellulosic biopolymers. A suitable cellulosic material is microbialcellulose. According to embodiments, the textile may include one or morecellulosic material.

According to embodiments, in addition to the cellulosic material, thetextile may comprise other materials (e.g., fibers) of natural origin,such as silk, wool, chitin, chitosan, and mixtures thereof.

According to embodiments, the textile may comprise materials (e.g.,fibers) of synthetic origin, such as polyester, nylon, polyurethane,spandex (elastane), acrylic, modacrylic, acetate, polyolefin, vinyl andmixtures thereof.

As above mentioned, according to embodiments, the textile may be one ormore textile selected from the group consisting of fiber(s), yarn(s),rope(s), fabric(s) and garment(s).

Suitable yarns may be manufactured by any known method, and suitablefabrics also may be manufactured by any known method, such as weaving,knitting, crocheting, knotting, and felting. The fabric may be anon-woven fabric. Furthermore, suitable garments may be any garment,such as jeans, shirts, casual wear garments, etc.; suitable garments mayinclude woven denim fabrics.

According to embodiments, the textile may be a yarn or a fabric.According to embodiments, the textile is a fabric, preferably a wovenfabric, more preferably a denim fabric.

As above mentioned, the process of the invention comprises a step a) ofproviding indole or an indole derivative to at least part of saidtextile.

According to the present invention, “indole derivatives”, “indoxylderivatives” and “indigo derivatives” refer to respectively indole,indoxyl and indigo substituted by one or more substituents, for examplesubstituted by: one or more groups on one or more carbons in anyposition selected from positions 4, 5, 6 and 7 of indole or indoxyl, andfrom positions 4, 4′, 5, 5′, 6, 6′, 7, and 7′ of indigo, and/or by agroup on the nitrogen atom(s) of indole, indoxyl or indigo. The one ormore groups substituting one or more carbons may be groups such as, butnot limited to, halogen groups, alkyl groups, alkoxy groups, arylgroups, aryloxy groups, amine groups, nitro groups and carbonyl groups.The group substituting nitrogen atom(s) may be groups such as, but notlimited to, alkyl groups, aryl groups, and acyl groups. Indolederivatives may be, for example, 4-chloroindole, 5-chloroindole,6-chloroindole, 7-chloroindole, 5-bromoindole, 6-bromoindole,5-nitroindole, 5-hydroxyindole, 5-methylindole, 5-methoxyindole,6-methylindole, 7-methylindole, 5-aminoindole, 1-methylindole,indole-6-carboxaldehyde; and indoxyl derivatives can be, for example,4-chloroindoxyl, 5-chloroindoxyl, 6-chloroindoxyl, 7-chloroindoxyl,5-bromoindoxyl, 6-bromoindoxyl, 5-nitroindoxyl, 5-hydroxyindoxyl,5-methylindoxyl, 5-methoxyindoxyl, 6-methylindoxyl, 7-methylindoxyl,5-aminoindoxyl, 1-methylindoxyl, indoxyl-6-carboxaldehyde. It isencompassed in the present invention also the use of any other indoleand indoxyl derivatives, provided that such indole derivatives can bereacted and converted into the correspondent indoxyl derivatives byenzymatic catalysis. These indoxyl derivatives, when converted (e.g.,dimerized) provide the correspondent indigo derivatives, which have eacha different color. According to the present invention, the term “indigoderivatives” refers also to asymmetric indigo, i.e. indigo deriving fromdimerization of two different indoxyl derivatives, or of indoxyl and anindoxyl derivative. Dyeing of the textile with asymmetric indigo can beachieved according to the process of the invention when, for example, asolution comprising two or more different indole derivatives, or indoleand one or more indole derivatives, are used. As used herein, the term“indigo derivatives” refer also to asymmetric indigo, i.e. indigoderiving from dimerization of two different indoxyl derivatives, or ofindoxyl and an indoxyl derivative. Dyeing of the textile with asymmetricindigo can be achieved according to the process of the invention whentwo or more different indole derivatives, or indole and one or moreindole derivatives, are used. For example, when two different indolederivatives, or indole and an indole derivative, are used, two differentindoxyl derivatives, or indoxyl and an indoxyl derivative, are obtained.Advantageously, when such two different indoxyl derivatives, or indoxyland an indoxyl derivative, are used, three different indigo derivativesare obtained (namely, two different symmetric indigo derivatives and anasymmetric indigo derivative), so that a textile can be dyed with morethan one dye, in particular, by converting such two different indoxylderivatives, or indoxyl and an indoxyl derivative, to indigoderivatives, on the textile, whereby providing the dye onto the textile.

Indole can be provided to the textile according to methods that areknown, per se, in the art, such as dipping and spraying, so that thetextile is provided with indole. For example, indole can be applied tothe textile according to techniques that are known in the art to besuitable to provide leuco indigo to textiles, such as, for example, ropedyeing, slasher dyeing, loop dyeing and continuous fabric dyeingtechniques. In this case, at least step a) of the process of theinvention may be carried out according to techniques that are well knownin the art, using indole instead of leuco indigo.

Without being bound to a specific scientific explanation, when indole isprovided to the textile, indole is adsorbed in the fibers of thetextile. In this case, the use of an oxidizing hybrid enzyme including acellulose-binding domain (CBD) allows to effectively obtain aring-dyeing effect on the textile. Without being bound to a specificscientific explanation, this effect may derive from an effectiveconversion on indole to indigo occurring on the surface of the textile,due to the presence of the hybrid oxidizing enzyme. Also, when, inparticular, dye precursors that can be negatively charged are used(e.g., tryptophan or derivative thereof) the depth of penetration ofsuch dye precursors between the fibers of the textiles can be varied, sothat different dyeing effects (e.g., ring dyeing effects) can beobtained.

According to an aspect, the process of the invention comprises a step b)of providing at least an oxidizing enzyme to at least part of thetextile, whereby said at least part of textile includes said indole orindole derivative and said oxidizing enzyme, wherein the oxidizingenzyme is an oxidizing hybrid enzyme comprising a cellulose bindingdomain (CBD).

As used herein, the term “hybrid enzyme” refers to an enzyme which havebeen genetically modified to include a cellulose binding domain (CBD) ora collagen-binding domain, or a chitin-binding domain, or achitosan-binding domain, or a domain suitable to bind syntheticpolymeric materials or fibers, e.g., a polyester-binding domain. Anenzyme, e.g., an oxidizing enzyme, may be modified to include a bindingdomain suitable for binding a material which is included into thetextile to be dyed, and/or for increasing affinity for binding amaterial in the textile, with respect to the unmodified enzyme. Forexample, an enzyme that has been genetically modified to include a CBDhas an increased affinity for binding cellulosic materials, with respectto the unmodified enzyme. Additionally, it is known thatcellulose-binding domains (CBDs) can also bind to chitin or chitosan.

As used herein, the term “oxidizing hybrid enzyme” refers to anoxidizing enzyme which has been genetically modified to include acellulose binding domain (CBD) or a collagen-binding domain, or achitin-binding domain, or a chitosan-binding domain, or a domainsuitable to bind synthetic polymeric materials or fibers that arepresent in the yarn to be dyed, e.g., a polyester-binding domain.

As used herein, “oxidizing enzyme” refers to an enzyme that is able tocatalyze oxidation of its substrates, such as an oxidoreductase (EC 1).Suitable oxidoreductase is a monooxygenase (EC 1.13); it preferably is aflavin-containing monooxygenase (FMOs) (EC 1.14.13.8), and morepreferably a microbial flavin-containing monooxygenase (mFMO).Alternatively, the monooxygenase can be a Baeyer-Villiger monooxygenase(BVMO). Monooxygenases, in particular FMOs and mFMOs, provide goodconversion rates and binding of many dye precursors, such as indoleand/or derivatives thereof, as well as a suitable specificity to convertindole derivatives, and are thus suitable to be used in the process ofthe invention. Baeyer-Villiger monooxygenases (BVMOs) have closehomology to FMOs, and are thus suitable as well to be used in theinvention. A suitable oxidizing enzyme to be used in the invention ismFMO from Methylophaga sp., more preferably from the strain SK1.

As used herein, the term “oxidizing enzyme” also encompasses geneticallymodified oxidizing enzymes that have been genetically modified toimprove the enzyme's properties, such as oxidation efficiency of thesubstrate(s) of the oxidizing enzyme, for example, by changing one ormore amino acid residues.

The oxidizing hybrid enzyme may be provided to the textile according tomethods that are known, per se, in the art. According to embodiments,the oxidizing hybrid enzyme is provided to the textile by spraying orpouring, preferably by spraying.

As above mentioned, the process of the invention comprises a step c) ofconverting at least part of the indole or indole derivative, to indigoor indigo derivative.

Without being bound to a specific scientific explanation, it is believedthat oxidizing enzymes, suitable to be used in the process of theinvention, catalyze the hydroxylation of indole and/or indolederivative(s), to provide indoxyl and/or the corresponding indoxylderivative(s), that eventually dimerize to indigo, and indigoderivatives, respectively.

According to an aspect of the invention, the oxidizing enzyme is ahybrid enzyme comprising a cellulose-binding domain (CBD).

As used herein, a typical cellulose-binding domain (CBD) may be onewhich occurs in a cellulase and which binds preferentially to celluloseand/or to poly- or oligosaccharide fragments thereof. Additionally, itis known that cellulose-binding domains (CBDs) can bind also to chitinand chitosan.

Cellulose binding domains and (hybrid) enzymes including such domainsare known, per se in the art, for example, from document WO97/28256 anddocument WO97/40229. Cellulose-binding domains are polypeptide aminoacid sequences which occur as integral parts of polypeptides or proteinsconsisting of two or more polypeptide amino acid sequence regions, forexample in hydrolytic enzymes (hydrolases), which typically comprise acatalytic domain containing the active site for substrate hydrolysis anda cellulose-binding domain for binding to the cellulosic substrate inquestion. Such enzymes can comprise more than one catalytic domain andone, two or three cellulose-binding domains, and they may furthercomprise one or more polypeptide amino acid sequence regions linking thecellulose-binding domain(s) with the catalytic domain(s), a region ofthe latter type usually being denoted a “linker”. Enzymes comprising acellulose-binding domain are known, per se, in the art. Examples ofhydrolytic enzymes comprising a cellulose-binding domain are cellulases,xylanases, mannanases, arabinofuranosidases, acetylesterases andchitinases. “Cellulose-binding domains” have also been found in algae,e.g. in the red alga Porphyra purpurea in the form of a nonhydrolyticpolysaccharide-binding protein [see P. Tomme et al., Cellulose-BindingDomains—Classification and Properties, in: Enzymatic Degradation ofInsoluble Carbohydrates, John N. Saddler and Michael H. Penner (Eds.),ACS Symposium Series, No. 618 (1996)]. However, most of the known CBDs[which are classified and referred to by P. Tomme et al. (op cit.) as“cellulose-binding domains”1 derive from cellulases and xylanases. TheP. Tomme et al. reference classifies more than 120 “cellulose-bindingdomains” into 10 families (I-X) which may have different functions orroles in connection with the mechanism of substrate binding. Inproteins/polypeptides in which CBDs occur (e.g. enzymes, typicallyhydrolytic enzymes such as cellulases), a CBD may be located at the N orC terminus or at an internal position. That part of a polypeptide orprotein (e.g. hydrolytic enzyme) which constitutes a CBD per setypically consists of more than about 30 and less than about 250 aminoacid residues. For example: those CBDs listed and classified in Family Iin accordance with P. Tomme et al. (op. cit.) consist of 33-37 aminoacid residues, those listed and classified in Family IIa consist of95-108 amino acid residues, those listed and classified in Family VIconsist of 85-92 amino acid residues, whilst one CBD (derived from acellulase from Clostridium thermocellum) listed and classified in FamilyVII consists of 240 amino acid residues. Accordingly, the molecularweight of an amino acid sequence constituting a CBD per se willtypically be in the range of from about 4 kD to about 40kD, and usuallybelow about 35kD.

In general, modified enzymes (i.e., hybrid enzymes) including acellulose binding domain, as well as detailed descriptions of thepreparation and purification thereof, are known, per se, in the art[see, e.g., WO 90/00609, WO 94/24158 and WO 95/16782, as well asGreenwood et al., Biotechnology and Bioengineering 44 (1994) pp.1295-13051. They may, e.g., be prepared by transforming into a host cella DNA construct comprising at least a fragment of DNA encoding thecellulose-binding domain ligated, with or without a linker, to a DNAsequence encoding the enzyme of interest (in the present case, anoxidizing enzyme), and growing the transformed host cell to express thefused gene.

According to embodiments, type of recombinant product (i.e., enzymehybrid) obtainable in this manner—often referred to in the art as afusion protein—may be described by one of the following generalformulae:

A-CBD-MR-X—B

A-X-MR-CBD-B

In the above formulae, CBD is an amino acid sequence comprising at leastthe cellulose-binding domain (CBD) per se.

MR (the middle region; a linker) may be a bond, or a linking groupcomprising from 1 to about 100 amino acid residues, in particular offrom 2 to 40 amino acid residues, e.g. from 2 to 15 amino acid residues.MR may, in principle, alternatively be a non-amino-acid linker.

X is an amino acid sequence comprising at least the catalytically(enzymatically) active sequence of amino acid residues of an enzyme,e.g., an oxidizing enzyme, as above defined, encoded by a DNA sequenceencoding the enzyme of interest.

The moieties A and B are independently optional. When present, a moietyA or B constitutes a terminal extension of a CBD or X moiety, andnormally comprises one or more amino acid residues.

According to embodiments, the CBD in the genetically modified enzymeaccording to the present invention, may be positioned C-terminally,N-terminally or internally in the enzyme hybrid, i.e., in thegenetically modified enzyme.

Hybrid enzyme of interest in the context of the invention include enzymehybrids which comprise more than one CBD, e.g. such that two or moreCBDs are linked directly to each other, or are separated from oneanother by means of spacer or linker sequences (consisting typically ofa sequence of amino acid residues of appropriate length). Two CBDs in anenzyme hybrid of the type in question may, for example, also beseparated from one another by means of an -MR-X— moiety as definedabove.

According to embodiments, the hybrid enzyme may be described by thefollowing formula:

CBD-MR—X

wherein:

CBD can be either the N-terminal or the C-terminal region of an aminoacid sequence corresponding to at least the cellulose-binding domain; MRis the middle region (the linker), and may be a bond, or a short linkinggroup of from about 2 to about 100 carbon atoms, in particular of from 2to 40 carbon atoms, or typically from about 2 to about 100 amino acids,in particular of from 2 to 40 amino acids, and X can be either theN-terminal or the C-terminal region and is an enzyme, for example anoxidizing enzyme, e.g., a monooxygenase.

The above discussion made with reference to cellulose-binding domain(CBD) applies mutatis mutandis to collagen-binding domains, orchitin-binding domains, or chitosan-binding domains, or domains thatbind to synthetic polymeric materials, e.g., polyester-binding domains.

Collagen-binding domains and proteins containing collagen-bindingdomains are known, per se, in the art. For example, a suitablecollagen-binding domain may be derived from the collagenase ofClostridium histolyticum and it is disclosed in Nishi et al.,“Collagen-binding growth factors: Production and characterization offunctional fusion proteins having a collagen-binding domain”, Proc.Natl. Acad. Sci. USA; Vol. 95, pp. 7018-7023, June 1998.Collagen-binding domains suitable to be used in the present inventionare also disclosed in EP2940041A1 (see, e.g., par. [0024]). For example,collagen-binding domains suitable to be used in the present inventionare may be derived, from fibronectin, collagenase, integrin al chain,integrin α2 chain, integrin α10 chain, integrin all chain, plateletglycoprotein VI, discoidin domain receptor 1, discoidin receptor 2,mannose receptor, phospholipase A2 receptor, von Willebrand factor,leukocyte-associated immunoglobulin-like receptor 1 andleukocyte-associated immunoglobulin-like receptor 2.

Chitin-binding domains and proteins containing chitin-binding domainsare known, per se, in the art. For example, chitin-binding domainssuitable to be used in the present invention are disclosed inEP2599790A1 (see, e.g., par. [102], Table 6 and Table 7). For example,chitin-binding domains suitable to be used in the present invention maybe derived, for example, from chitinases, chitobiases and chitin-bindingproteins.

Chitosan-binding domains and proteins containing chitosan-bindingdomains are known, per se, in the art. For example, chitosan-bindingdomains suitable to be used in the present invention are disclosed inEP2599790A1 (see, e.g., par. [102]) and in Chen, HP; Xu, LL, (2005) J.of Integrative Plant Biology 47(4): 452-456.

Domains that bind to synthetic polymeric materials, e.g.,polyester-binding domains are known, per se, in the art. In embodiments,the domains that bind to synthetic materials may be in the form ofanchor peptides. Domains that bind to synthetic polymeric materials thatare suitable to be used according to the present invention are, forexample, those disclosed in Islam et al., “Targeting microplasticparticles in the void of diluted suspentions”, EnvironmentalInternational 123 (2019) 428-435. For example, domains that bind tosynthetic materials suitable to be used in the present invention may bethe anchor peptide LC1 and/or the anchor peptide Tachystatin A2 (TA2).

As above disclosed with reference to modified enzymes (i.e., hybridenzymes) including a cellulose binding domain, modified enzymes (i.e.,hybrid enzymes) including a collagen binding domains, chitin bindingdomains, chitosan binding domains and domains that bind to syntheticpolymeric materials, may be produced and purified through techniquesthat are, per se, known in the art. For example, such hybrid enzymes maybe prepared by transforming into a host cell a DNA construct comprisingat least a fragment of DNA encoding the selected binding domain ligated,with or without a linker, to a DNA sequence encoding the enzyme ofinterest (in the present case, an oxidizing enzyme), and growing thetransformed host cell to express the fused gene. Purification of theexpressed hybrid enzyme may be carried out through techniques that are,per se, known in the art.

According to embodiments, the textile can include, in addition to thecellulosic material or as an alternative to the cellulosic material,collagen, chitin and/or chitosan, and synthetic materials, e.g.,polyester. In this case, the oxidizing hybrid enzyme may include, inaddition to the cellulose-binding domain (CBD) or in alternative to CBD,a collagen-binding domain, chitin-binding domain, a chitosan bindingdomain or a domain that bind to such synthetic materials, e.g., apolyester-binding domain. According to embodiments, when a tryptophanaseis used, the tryptophanase may be a tryptophanase hybrid enzymeincluding, in addition to a CBD or in alternative to a CBD, acollagen-binding domain, chitin-binding domain, a chitosan bindingdomain or a domain that bind to such synthetic polymeric materials,e.g., a polyester-binding domain.

According to embodiments, in the process of the invention, the textileis provided with indole or indole derivative, i.e., an indole(derivative) is applied to the textile in an amount suitable to impart adyeing effect. The textile treated with indole is then provided with theoxidizing hybrid enzyme. In other words, according to preferredembodiments, step a) of the process is carried out before step b).Accordingly, the textile is treated with indole and, subsequently,treated with the hybrid oxidizing enzyme. In this case, it has beenobserved that indole penetrates between the fibers of the textile. Oncethe oxidizing enzyme including a CBD is applied, it will convert indoleto indigo, thereby obtaining a particularly effective dyeing of thetextile, e.g., a particularly effective ring dyeing.

As above mentioned, according to embodiments, indole can be provided tothe textile by conventional methods, e.g., dipping or spraying, andsubsequently, the oxidizing hybrid enzyme may be provided by spraying.Subsequently, according to embodiments, the textile may be exposed toair.

According to embodiments, in the process of the invention, the textileis provided with the oxidizing hybrid enzyme and then provided withindole or indole derivative. In other words, according to embodiments,step b) of the process is carried out before step a). For example, theoxidizing hybrid enzymes may be provided to the textile by spraying andthen the textile may be dipped in a bath including indole, e.g., areaction mixture including indole. In this case, it has been observedthat the oxidizing hybrid enzymes have high affinity for the cellulosicmaterial of the textile through its cellulose-binding domain (CBD),whereby the enzymatic conversion of indole (or an indole derivative) toindoxyl (or indoxyl derivative) and the subsequent non-enzymaticconversion of indoxyl (or indoxyl derivative) to indigo (or indigoderivative) occurs on the textile, so that substantially all the indigoobtained is provided to the textile.

According to embodiments, the process of the invention may include astep of opening the fibers of the textile during step a) of providingindole and/or during step b) of providing the enzyme. Fibers of atextile can be opened, e.g., by increasing the space between a fiber andanother, according to techniques that are known, per se, in the art. Inthis case, penetration of indole and/or of the enzymes is particularlyeffective.

According to embodiments, at least part of said indole or indolederivative and/or at least part of said indigo and/or indigo derivativeis located between the fibers of said textile.

Oxidizing enzymes require O₂, i.e. oxygen, in order to catalyze thehydroxylation of indole or its derivative to indoxyl or indoxylderivatives. The O₂ required, can be the oxygen normally dissolvedwithin, for example, in the aqueous indole solution, and/or in theaqueous mixture including the oxidizing enzyme.

Conversion of indoxyl to indigo can occur spontaneously, when, forexample, O₂ concentration is in an adequate amount to oxidize indoxyl orits derivatives. The O₂ required, can be the atmosphere oxygen and/orthe oxygen normally dissolved within, for example, the indole solution,and/or in the aqueous mixture including the oxidizing enzyme.

According to embodiments, step c) of converting at least part of theindole to indigo is carried out in presence of oxygen (O₂), for example,exposing the textile to oxygen, e.g., to air.

According to embodiments, at least step b) of providing the enzymeand/or step c) of converting indole to indigo may be carried out ininert or substantially inert atmosphere, e.g., in an atmosphere withreduced amount of oxygen to delay the conversion of indoxyl to indigo.

After the textile is provided with indigo or a derivative thereof, itmay be washed and/or rinsed, e.g., to remove the oxidizing hybrid enzymefrom the cellulosic material, e.g., from cotton fibers, and dried.

Removal of the enzyme from the textile may be carried out by changing pHand/or temperature. Indeed, an enzyme that is immobilized to, forexample, a cellulosic material (e.g., cotton) through a CBD, can remainon the cellulosic material for several days (e.g., 7-10 days), when thepH is in the range between about 3 and about 8, and/or the temperatureis between about 4° C. to about 50° C.

Thus, a change of pH and/or temperature can be applied to remove thehybrid enzyme from the cellulosic material, e.g., raising the pH (forexample, on the surface of the fabric) above 8, and or raising thetemperature above 50° C.

According to embodiments, the process further comprises a step d) ofremoving at least the oxidizing hybrid enzyme from the textile,preferably by washing and/or rinsing the textile at a pH lower thanabout 3 or higher than about 8 and/or to a temperature lower than about4° C. or higher than about 50° C.

Suitable oxidizing enzymes are oxygenases that are capable tohydroxylate indole or indole derivatives to obtain indoxyl or indoxylderivatives. According to embodiments, the oxidizing enzyme is anoxygenase, preferably a monooxygenase, more preferably is aflavin-containing monooxygenases (FMO).

According to embodiments, the oxidizing hybrid enzyme is CBD-FMO,preferably CBD-mFMO.

According to embodiments, a textile including a cellulosic material isprovided with indole or an indole derivative, at least an oxidizinghybrid enzyme, suitable cofactors, and optionally one or more cofactorregenerating enzymes.

According to embodiments, the aqueous mixture including the enzyme(s)may comprise functional solutes, such as salts, buffering agents andoxygen and/or peroxide scavengers (e.g. catalases). Catalase may beprovided to convert possibly formed H₂O₂ into O₂ and H₂O.

According to embodiments, indole is applied to the textile as an indoleprecursor. Namely, according to embodiments, step a) of the process ofthe invention comprises a step of providing tryptophan or a tryptophanderivative, and at least a tryptophanase, to the textile, and convertingsaid tryptophan or tryptophan derivative to obtain the indole or indolederivative. When tryptophan is used, a better ring-dyeing effect can beobtained, in particular when the textile includes cellulosic materials.Without being bound to a specific scientific explanation, this effectmay derive from the fact that, in view of their properties, tryptophanand tryptophan derivatives, when provided to the textile, will bepredominantly localized on the surface of the textile (in particularwhen the textile includes at least a cellulosic material), thusproviding an improved ring effect.

In other words, tryptophan and/or a tryptophan derivative can beprovided to the textile instead of, or in addition to, indole or indolederivatives. In this case, at least a tryptophanase may be provided tothe textile, in addition to the oxidizing hybrid enzyme, to convert atleast part of the tryptophan to indole. According to embodiments,tryptophan and/or a tryptophan derivative can be used as startingmaterial (i.e., starting substrate) in the process of the invention, toenzymatically produce indole or indole derivatives. Accordingly,tryptophan and/or a tryptophan derivative can be used as startingmaterials (i.e., starting substrates) to obtain indigo and/or indigoderivatives, through enzymatic reactions.

Tryptophanases (systematic name: L-tryptophan indole-lyase (deaminating;pyruvate-forming)) are enzymes, per se known, that cleave acarbon-carbon bond of tryptophan, releasing indole. They may usepyridoxal phosphate (PLP) as cofactor. According to embodiments of theinvention, PLP can be optionally used to improve the yield of theenzymatic conversion of tryptophan or of its derivatives catalyzed bytryptophanase. Tryptophanases suitable to be used in the process of theinvention are known in the art. For example, a tryptophanase suitable tobe used in the method of the invention is the tryptophanase ofEscherichia coli NEB® 10β.

As used herein, the term “tryptophan derivative” refers to tryptophansubstituted with one or more substituents, as above disclosed, mutatismutandis, with reference to indole, indoxyl and indigo derivatives. Forexample, a tryptophan derivative may be a halogenated derivative oftryptophan, i.e., halogenated tryptophan (e.g., 6-bromotryptophan).

According to embodiments, the tryptophan derivative is a6-bromotryptophan (i.e., a halogenated tryptophan) and the indigoderivative is Tyrian purple.

As used herein, the term “halogenated derivative” refers to anytryptophan, indole, indoxyl and indigo substituted with a halogen atom,in particular fluorine, chlorine, bromine or iodine atom, on one or morecarbons in position 5, 6, 7 and 8 (and also 5′, 6′, 7′ and 8′ forindigo).

According to embodiments, the textile may be provided with tryptophan ora tryptophan derivative, a tryptophanase, an oxidizing hybrid enzyme,suitable cofactors, and optionally one or more cofactor regeneratingenzymes. Tryptophan is enzymatically converted to indole by thetryptophanase. Indole is converted to indoxyl by the oxidizing hybridenzyme. Indoxyl is (non-enzymatically) converted (e.g., dimerized) toindigo, to dye the textile.

As above discussed with reference to indole, tryptophan can be providedto the textile according to methods that are known, per se, in the art,such as dipping and spraying. Suitable techniques for providingtryptophan to the textile are techniques that are known in the art to besuitable to provide leuco indigo to textiles, such as, for example, ropedyeing, slasher dyeing, loop dyeing and continuous fabric dyeingtechniques. In this case, at least the step of providing tryptophan tothe textile of the process of the invention, may be carried outaccording to techniques that are well known in the art for impregnatingtextiles with leuco indigo, but using tryptophan instead of leucoindigo.

According to embodiments, the textile (e.g., a yarn or a fabric) may betreated, e.g., impregnated, with tryptophan and subsequently providedwith at least a tryptophanase (e.g., by spraying), to convert tryptophanto indole. Subsequently, the textile may be provided with the oxidizinghybrid enzyme (e.g., by spraying) to convert indole to indoxyl to obtainindigo.

In other words, according to embodiments, tryptophanase may be providedto the textile before the oxidizing hybrid enzyme. In this case,according to embodiments, tryptophanase is preferably not removed fromthe textile before providing the oxidizing enzyme.

According to embodiments, at least part of the oxidizing hybrid enzymeis provided to the textile together with said tryptophanase.

In other words, according to embodiments, the textile may be treated,e.g., impregnated, with tryptophan and subsequently provided (e.g., byspraying) with a mixture of at least a tryptophanase and at least anoxidizing hybrid enzyme, thereby converting tryptophan to indigo.

According to embodiments, the tryptophanase is a tryptophanase hybridenzyme comprising a cellulose-binding domain (CBD).

According to embodiments, enzymes may be removed from the textile bywashing and/or rinsing, preferably at a pH lower than about 3 or higherthan about 8 and/or to a temperature lower than about 4° C. or higherthan about 50° C.

One or more of the enzymes used in the process of the invention mayrequire one or more cofactors.

As used herein, the term “cofactor” refers to a non-protein chemicalcompound that is required for an enzyme's activity as a catalyst.Cofactors can be divided into two types, either inorganic ions, orcomplex organic molecules called coenzymes. For sake of clarity, in thepresent description, the term “cofactor” is used to indicate anynon-protein chemical compound that is required for an enzyme's activity,according to the protein of the invention, without restriction to aspecific chemical class of molecules, i.e., including both organic andinorganic molecules.

According to embodiments, cofactor regenerating enzymes may be used toregenerate the cofactor(s) which may be needed by the enzymes used inthe process of the invention.

In this case, advantageously, expensive cofactors (e.g., NADPH) areregenerated by consuming inexpensive cofactors (such as glucose,phosphite or formate).

For example, oxidizing enzymes such as FMOs may use NADPH as cofactorwhich may be produced by the NADPH regenerating enzyme that uses cheapcofactors such as glucose, phosphite and formate.

According to embodiments, the step of hydroxylating indole (or an indolederivative) in the presence of at least an oxidizing hybrid enzyme, toobtain indoxyl or an indoxyl derivative, may be carried out in thepresence of at least an enzyme suitable for regenerating the cofactor,required by the oxidizing enzyme. For example, when the oxidizing enzymeis a monooxygenase, NADPH may be used as cofactor.

According to embodiments, the oxidizing enzyme may be coupled to acofactor-regenerating enzyme, preferably fused to acofactor-regenerating enzyme.

In other words, according to embodiments, the oxidizing enzyme may be afusion enzyme wherein the oxidizing enzyme is fused to acofactor-regenerating enzyme. In this case, the oxidizing hybrid enzymecomprising a cellulose-binding domain is fused to acofactor-regenerating enzyme, i.e., further comprises acofactor-regenerating enzyme. For example, if the oxidizing enzyme is amicrobial flavin-containing monooxygenase (mFMO), and thecofactor-regenerating enzyme is phosphite dehydrogenase (PTDH), theoxidizing hybrid enzyme may be described by the following formula:

CBD-MR-PTDH-mFMO

wherein

CBD can be either the N-terminal or the C-terminal region of an aminoacid sequence corresponding to at least the cellulose-binding domain; MRis the middle region (the linker), and may be a bond, or a short linkinggroup of from about 2 to about 100 carbon atoms, in particular of from 2to 40 carbon atoms, or typically from about 2 to about 100 amino acids,in particular of from 2 to 40 amino acids, PTDH is thecofactor-regenerating enzyme, and mFMO is the monooxygenase, and can beeither the N-terminal or the C-terminal region.

According to embodiments, the cofactor-regenerating enzyme is selectedfrom the group consisting of glucose dehydrogenase (GDH), phosphitedehydrogenase (PTDH), and formate dehydrogenase (FDH), and preferably isPTDH. In embodiments, the cofactor-regenerating enzyme is suitable toregenerate NADPH and/or NADH cofactor.

According to embodiments, the cofactor regenerating enzyme can be amutant that has improved activity. For example, the NADPH regeneratingenzyme can be a mutant that has improved NADPH production, e.g. PTDHdisclosed in WO 2004/108912 A2.

According to embodiments, when, for example, the oxidizing enzyme ismFMO and the cofactor-regenerating enzyme is PTDH, the hybrid oxidizingenzyme may include the fusion enzyme PTDH-mFMO, wherein mFMO and/or PTDHhave been genetically modified to include a cellulose binding domain(CBD).

According to embodiments, the oxidizing hybrid enzyme comprises anoxidase, a cellulose binding domain, and further comprises a cofactorregenerating enzyme, preferably PTDH.

According to embodiments of the process of the invention, indole orindole derivatives may be obtained by converting tryptophan, or atryptophan derivative, in the presence of a tryptophanase, and PLP maybe used as cofactor in the reaction catalyzed by the tryptophanase.

As above discussed, an enzyme, e.g., an oxidizing enzyme, may bemodified to include a binding domain suitable for binding a materialwhich is included into the textile to be dyed, and/or for increasingaffinity for binding a material in the textile, with respect to theunmodified enzyme. For example, it has been observed that an enzyme thathas been genetically modified to include a CBD has an increased affinityfor binding cellulosic materials, with respect to the unmodified enzyme.Additionally, it has also be observed that cellulose-binding domains(CBDs) can also bind to chitin or chitosan. According to embodiments, ahybrid enzyme, e.g., an oxydizing hybrid enzyme and/or a tryptophanasehybrid enzyme may include a cellulose binding domain (CBD) or acollagen-binding domain, or a chitin-binding domain, or achitosan-binding domain, or a domain suitable to bind syntheticpolymeric materials or fibers, e.g., a polyester-binding domain.

An object of the present is therefore a process for dyeing a textileincluding collagen, comprising the following steps:

-   -   a) providing indole or an indole derivative to at least part of        the textile;    -   b) providing at least an oxidizing enzyme to at least part of        the textile, whereby said at least part of textile includes said        indole or indole derivative and said oxidizing enzyme;    -   c) converting at least part of the indole or indole derivative        to indigo or indigo derivative, thereby dyeing at least part of        the textile;        wherein said oxidizing enzyme is an oxidizing hybrid enzyme        comprising a collagen-binding domain.

Also, an object of the present invention is a process for dyeing atextile including chitin and/or chitosan, comprising the followingsteps:

-   -   a) providing indole or an indole derivative to at least part of        the textile;    -   b) providing at least an oxidizing enzyme to at least part of        said textile, whereby said at least part of textile includes        said indole or indole derivative and said oxidizing enzyme;    -   c) converting at least part of the indole or indole derivative        to indigo or indigo derivative, thereby dyeing at least part of        the textile;        wherein said oxidizing enzyme is an oxidizing hybrid enzyme        comprising a chitin-binding domain or a chitosan binding domain        or a cellulose binding domain (CBD).

A further object of the present invention is a process for dyeing atextile including a synthetic polymeric material, preferably polyester,comprising the following steps:

-   -   a) providing indole or an indole derivative to at least part of        the textile;    -   b) providing at least an oxidizing enzyme to at least part of        said textile, whereby said at least part of textile includes        said indole or indole derivative and said oxidizing enzyme;    -   c) converting at least part of the indole or indole derivative        to indigo or indigo derivative, thereby dyeing at least part of        said textile;        wherein said oxidizing enzyme is an oxidizing hybrid enzyme        comprising a synthetic polymeric material-binding domain,        preferably a polyester-binding domain.

The above discussion made with reference to the process for dyeingtextiles including a cellulosic material, also applies to theabove-mentioned processes for dyeing textiles including collagen, chitinand/or chitosan, and a synthetic polymeric material, preferablypolyester.

Another object of the present invention is a dyed textile as obtainableaccording to the process of the invention.

According to embodiments, the textile is a yarn or a plurality of yarns,such as, for example, a rope.

According to embodiments, the textile is a fabric, preferably a wovenfabric, more preferably a twill fabric.

According to embodiments, the textile obtained through the process ofthe invention is indigo dyed.

A further object of the present invention is an oxidizing hybrid enzyme,namely a modified enzyme comprising at least an oxidizing enzyme,preferably an oxygenase, a cellulose binding domain or acollagen-binding domain, or a chitin-binding domain, or a chitosanbinding domain, or a synthetic polymeric material-binding domain.

An exemplary synthetic material binding domain is polyester bindingdomain.

According to embodiments, the hybrid enzyme further comprises acofactor-regenerating enzyme.

According to embodiments, the oxygenase is a monooxygenase, preferablymFMO, and the cofactor regenerating enzyme is PTDH.

As above mentioned, it has been observed that the use of a modifiedoxidizing enzyme including a CBD according to the invention allows forproviding to the textile an increased amount of dye with respect to theuse of the unmodified oxidizing enzymes.

A further object of the invention is a tryptophanase hybrid enzymecomprising a tryptophanase, and a cellulose-binding domain (CBD) or acollagen-binding domain, or a chitin-binding domain, or a chitosanbinding domain, or a synthetic polymeric material-binding domain. Anexemplary synthetic material binding domain is polyester binding domain.

Binding domains suitable to bind to synthetic materials, e.g., syntheticmaterials different from polyester, may be designed and producedaccording to techniques that are known, per se, in the art.

The process of the invention provides the synthesis of indigo or anindigo derivative, optionally starting from tryptophan or a tryptophanderivative, by means of the combination of at least an enzymaticreaction step carried out by the oxidizing hybrid enzyme, and anon-enzymatic step. The process of the invention is particularlyadvantageous to provide indigo and/or indigo derivatives, such as Tyrianpurple, to textiles, whereby dyeing at least part of such textiles, in acost-effective way.

Methods for the preparation and the purification of hybrid enzymesaccording to the present invention, are known, per se, in the art.According to embodiments, the oxidizing hybrid enzyme and/or thetryptophanase and/or the tryptophan halogenase are isolated enzymes,preferably purified enzymes.

As used herein, the term “purified” refers to enzymes free from othercomponents from the organism from which the enzymes are derived.

According to embodiments, a textile including a cellulosic material isprovided with indole or an indole derivative and with a reactionmixture, comprising, in a suitable buffer, an oxidizing hybrid enzymeand suitable cofactors. Preferably, the textile is provided with indoleor indole derivatives and, subsequently, with an aqueous mixtureincluding the oxidizing hybrid enzyme. Preferably, the oxidizing hybridenzyme (e.g., an aqueous mixture including the oxidizing hybrid enzyme)is provided to the fabric by spraying or pouring.

According to preferred embodiments, the indole may be provided to the byspraying or pouring, or textile may be immersed in a mixture comprisingindole or an indole derivative. Subsequently the oxidizing hybrid enzymemay be sprayed or poured onto the textile.

According to other embodiments, the textile may be provided with amixture comprising the oxidizing hybrid enzyme (e.g., by spraying orpouring), and subsequently the textile may be provided with indole or anindole derivative.

According to embodiments, aqueous mixtures comprising indole and/orenzymes may include suitable buffers, suitable cofactors, functionalsolutes, such as salts, and oxygen and/or peroxide scavengers (e.g.catalases).

For example, the textile may be provided with indole and, subsequently,provided with a mixture including, in a suitable buffer, a monooxygenasehybrid enzyme, one or more cofactors, one or more cofactor regeneratingenzymes and optionally a catalase. In embodiments, the monooxygenasehybrid enzyme is fused with a cofactor regenerating enzyme, such as, forexample, CBD-linker-PTDH-mFMO.

According to the present invention, all the enzymes used in the processin the process of the invention may be genetically engineered.

Methods and processes for the production of genetically engineeredenzymes, as well as for the production of hybrid or fusion enzymes areknown, per se, in the art.

According to embodiments, indole and enzymes may be separately providedto the textile as aqueous mixtures. Such aqueous mixtures may have pHfrom about 5 to about 8, preferably from 5.5 to 8, more preferablybetween 6 and 7.5. Such aqueous medium may comprise a buffering agent,for example a potassium phosphate buffer.

According to embodiments, the process may be carried out at temperaturecomprised in the range from 20° C. to 50° C., preferably from 25° C. to40° C.

According to embodiments, the process may be carried out, for example,for a time that is sufficient to obtain a suitable amount of dye. Forexample, the process may per carried out for a time comprised between afew hours to several days, for example, 3-5 days.

In embodiments, temperature, pH values and duration of the process canvary and can be those conventionally used in enzymatic synthesis ofinsoluble dyes.

In embodiments, temperature, pH values and duration of the process, aswell as other parameters can be adjusted according to, for example,which type of textile has to be dyed and which dye is chosen as a finaldye.

A further object of the invention is a textile comprising an oxidizinghybrid enzyme and/or a tryptophanase hybrid enzyme.

According to embodiments, a textile comprising an oxidizing enzymeaccording to the invention may be undyed or at least in part dyed.

According to embodiments, a textile comprising a tryptophanase hybridenzyme may be undyed, in particular when the textile is not providedwith an oxidizing hybrid enzyme.

The present invention relates to a process wherein one or more dyeprecursors (e.g., tryptophan, indole or derivatives thereof) areprovided to a textile and converted by one or more enzymes (e.g., atryptophanase and an oxidizing enzyme) to provide the textile with adye, preferably indigo and/or one or more indigo derivatives, e.g., oneor more indigoid dyes.

In particular, the present invention relates to a process for dyeing atextile, comprising the following steps:

-   -   a) providing at least one dye precursor to at least part of said        textile;    -   b) providing one or more enzymes to at least part of said        textile, whereby said at least part of textile includes said dye        precursor and said enzyme;    -   c) converting at least part of said dye precursor to a dye,        thereby dyeing at least part of said textile;    -   wherein said one or more enzymes include an oxidizing hybrid        enzyme comprising a cellulose-binding domain (CBD).

According to an aspect, the textile includes a cellulosic material.According to embodiments, in addition to the cellulosic material or asan alternative to the cellulosic material, the textile may comprise oneor more of collagen, chitin, chitosan, and synthetic polymericmaterials, e.g., polyester. In this case, the oxidizing hybrid enzymemay include, a cellulose-binding domain (CBD) or a collagen-bindingdomain, or a chitin-binding domain, or a chitosan binding domain or adomain that bind to such synthetic polymeric materials, e.g., apolyester-binding domain.

According to embodiments, the dye precursor may be selected fromtryptophan, a tryptophan derivative, indole, an indole derivative, andmixtures thereof.

When the dye precursor includes tryptophan or a tryptophan derivative,said one or more enzymes include a tryptophanase.

In embodiments, the dye precursor may be provided to the textileaccording to methods that are known, per se, in the art, such as dippingand spraying, so that the textile is impregnated with the dye precursor.For example, the dye precursors can be provided according to techniquesthat are known in the art to be suitable to provide leuco indigo totextiles, such as, for example, rope dyeing, slasher dyeing, loop dyeingand continuous fabric dyeing techniques.

In embodiments, the enzymes may be provided to the textile according tomethods that are known, per se, in the art, e.g., dipping and spraying.According to embodiments, one or more enzymes may be provided to thetextile by spraying or pouring. Preferably, the oxidizing enzyme isprovided to the textile by spraying.

According to embodiments, the enzymes (e.g., aqueous mixtures comprisingenzymes), may be provided to a yarn or elongated element through theprocess discloses in the European patent application number EP3581705A1,in the name of the present applicant. Although EP3581705A1 relates to aprocess for providing a culture of microorganisms the process disclosedtherein may be applied, mutatis mutandis, to provide enzymes to yarns orelongated elements.

For example, an aqueous mixtures comprising enzymes suitable to be usedin the present invention, may be provided to a yarn, which has beenpreviously provided with a dye precursor, by means of an apparatuscomprising a feeding device having an outlet for dispensing such aqueousmixture from the outlet, and a yarn source to supply a yarn, that istreated with at least a dye precursor, to the feeding device, whereinthe apparatus is configured so that the aqueous mixture contacts atleast part of the yarn when the mixture is dispensed from the outlet.The dispensing of the mixture may be adjusted so that the mixture isdispensed from the outlet of the feeding device at a flow rate selectedso that the mixture envelops the yarn but is prevented from falling fromthe yarn, and from drying out at the outlet.

According to embodiments, the dye precursor is preferably provided tothe textile before the one or more enzymes.

According to embodiments, the process of the invention may include thefollowing steps:

-   -   a) providing tryptophan or a tryptophan derivative to at least        part of a textile;    -   b) providing at least a tryptophanase and at least an oxidizing        enzyme to at least part of said textile, whereby said at least        part of textile includes said tryptophan, said tryptophanase and        said oxidizing enzyme;    -   c) converting at least part of said tryptophan or tryptophan        derivative to indigo or indigo derivative, thereby dyeing at        least part of said textile;        wherein at least said oxidizing enzyme is a hybrid enzyme        comprising a cellulose-binding domain (CBD).

According to embodiments, when the dye precursor is tryptophan or atryptophan derivative, the tryptophanase is provided to the textilebefore the oxidizing hybrid enzyme. In this case, tryptophanase ispreferably not removed from the textile before providing the oxidizingenzyme.

According to embodiments, when the dye precursor is tryptophan or atryptophan derivative the tryptophanase and the oxidizing hybrid enzymesare provided together to the textile.

According to an aspect of the present invention, at least the oxidizingenzyme is a hybrid enzyme including a binding domain that is suitable tobind the enzyme to the textile and/or increase the affinity of theenzyme for the textile, namely to a material that is included into thetextile to be dyed, with respect to the unmodified enzyme. For example,an enzyme may be genetically modified to include a cellulose bindingdomain (CBD) or a collagen-binding domain, or a chitin-binding domain,or a chitos an-binding domain or a domain suitable to bind syntheticpolymeric materials or fibers, e.g., a polyester-binding domain. Forexample, when the textile to be dyed includes a cellulosic material, atleast the oxidizing enzyme is an oxidizing enzyme that has beengenetically modified to include a cellulose binding domain (CBD). Inembodiments, the tryptophanase may be a tryptophanase hybrid enzyme.

When the dye precursor is tryptophan or a tryptophan derivative, atleast part of a textile may be provided with said dye precursor andsubsequently provided with at least a tryptophanase and at least anoxidizing hybrid enzyme. Tryptophan is enzymatically converted to indoleby the tryptophanase, and indole is enzymatically converted to indoxylby the oxidizing enzyme. Indoxyl in non-enzymatically converted toindigo, thereby dyeing the textile.

When the dye precursor is indole or an indole derivative, at least partof a textile may be provided with said dye precursor and subsequentlyprovided with at least an oxidizing hybrid enzyme. Indole isenzymatically converted to indoxyl by the oxidizing enzyme, and indoxylin non-enzymatically converted to indigo, thereby dyeing the textile.

1. A process for dyeing a textile including a cellulosic material,comprising the following steps: a) providing indole or an indolederivative to at least part of said textile; b) providing at least anoxidizing enzyme to at least part of said textile, whereby said at leastpart of textile includes said indole or indole derivative and saidoxidizing enzyme; c) converting at least part of said indole or indolederivative to indigo or indigo derivative, thereby dyeing at least partof said textile; wherein said oxidizing enzyme is an oxidizing hybridenzyme comprising a cellulose-binding domain (CBD).
 2. The processaccording to claim 1, wherein said textile is provided with said indoleor indole derivative and then provided with said oxidizing hybridenzyme.
 3. The process according to claim 1, wherein said oxidizinghybrid enzyme is provided to said textile by spraying or pouring.
 4. Theprocess according to claim 1, wherein at least said step b) and/or saidstep c) are carried out in inert or substantially inert atmosphere. 5.The process according to claim 1, further comprising a step d) ofremoving at least said oxidizing hybrid enzyme from said textile, bywashing said textile at pH lower than 3 or higher than 8 and/or to atemperature lower than 4° C. or higher than 50° C.
 6. The processaccording to claim 1, wherein said cellulosic material is selected fromcotton, ramie, jute, flax, viscose, rayon, modal, lyocell, bamboo,microbial cellulose, and mixtures thereof.
 7. The process according toclaim 1, wherein said textile is selected from the group consisting of ayarn, a rope, a fabric and a garment.
 8. The process according to claim1, wherein at least part of said indole or indole derivative and/or atleast part of said indigo and/or indigo derivative is located betweenthe fibers of said textile.
 9. The process according to claim 1, whereinsaid oxidizing enzyme is an oxygenase.
 10. The process according toclaim 1, wherein said oxidizing hybrid enzyme is CBD-FMO.
 11. Theprocess according to claim 1, wherein said step a) comprises a step ofproviding tryptophan or a tryptophan derivative, and at least atryptophanase, to said textile, and converting said tryptophan ortryptophan derivative to obtain said indole or indole derivative. 12.The process according to claim 11, wherein at least part of saidoxidizing hybrid enzyme is provided to said textile together with saidtryptophanase.
 13. The process according to claim 11, wherein saidindole or indole derivative and/or said tryptophan or tryptophanderivative are provided to said textile by dipping, spraying, ropedyeing, slasher dyeing, loop dyeing or continuous fabric dyeing.
 14. Theprocess according to claim 11, wherein said tryptophanase is atryptophanase hybrid enzyme comprising a cellulose-binding domain (CBD).15. A process for dyeing a textile that includes one of collagen,chitin, chitosan, polyester, a synthetic polymeric material, comprisingthe following steps: a) providing indole or an indole derivative to atleast part of said textile; b) providing at least an oxidizing enzyme toat least part of said textile, whereby said at least part of textileincludes said indole or indole derivative and said oxidizing enzyme; c)converting at least part of said indole or indole derivative to indigoor indigo derivative, thereby dyeing at least part of said textile;wherein said oxidizing enzyme is an oxidizing hybrid enzyme comprising abinding domain selected from collagen-binding domain, chitin-bindingdomain, chitosan-binding domain, polyester-binding domain, a bindingdomain for a synthetic polymeric material.
 16. A dyed textile asobtainable through a process according to claim
 1. 17. A hybrid enzymecomprising an oxidizing enzyme or a tryptophanase enzyme, and at leastone binding domain selected from a cellulose-binding domain (CBD), acollagen-binding domain, a chitin-binding domain, a chitosan-bindingdomain, a polyester-binding domain, a synthetic polymericmaterial-binding domain.
 18. An oxidizing hybrid enzyme according toclaim 17, further comprising a cofactor regenerating enzyme, preferablyPTDH.
 19. A textile comprising a hybrid enzyme according to claim 17.